315 Proc. Pakistan Acad. Sci. 42(4):315-323.

2005
M. Zaheer Khan & F.C.P. Law

Review

ADVERSE EFFECTS OF PESTICIDES AND RELATED CHEMICALS ON

ENZYME AND HORMONE SYSTEMS OF FISH, AMPHIBIANS AND
REPTILES: A REVIEW

M. Zaheer Khan* and Francis C.P. Law

Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A IS6

Received February 2005, accepted October 2005

Communicated by Prof. Dr. M. A. Hafeez

Summary: The enzyme and hormone disrupting capabilities of pesticides and related chemicals are
suspected to be some of the factors contributing to the decline of fish, amphibian, and reptile
populations. Globally frogs and other amphibians have been disappearing at an alarming rate. In most
cases, the cause or causes are unknown, but are assumed to result from man-made pollutants in the
aquatic environment. Some current reports have indicated that many pesticides in the aquatic environment
are capable of disrupting the endocrine systems of animals. Some pesticides and related chemicals are
persistent in the environment and are accumulated in the fatty tissue of organisms and increase in
concentration as they move up through the food web. These chemicals are substances that can cause
adverse effects by interfering in some way with the body’s hormones or chemical messengers. A recent
study indicated that the atrazine effected the sexual development of frogs, even at extremely low doses.
Some pesticides reduce the cholinesterase activity of amphibians and reptiles. Some chemicals may
cause disease and reproductive failure in fish populations, because they bioaccumulate in the higher
trophic levels. Therefore, brown trout exposed to environmental pollutants have been shown to have
decreased egg size and low growth rate of the larvae.

Keywords: Pesticides, enzymes, hormones, amphibian, reptile, fish

Introduction mechanisms. Pesticides and related chemicals are

Pesticides and related chemicals destroy the
delicate balance between species that characterizes
a functioning ecosystem. Pesticides produce many
physiological and biochemical changes in freshwater
organisms by influencing the activities of several
enzymes. Alterations in the chemical composition
of the natural aquatic environment usually affect
behavioral and physiological systems of the
inhabitants, particularly those of the fish [1].
Hormone-disrupting effects in biota as a result
of chemicals are caused by a wide variety of
*To whom correspondence and reprint requests should be addressed.
substances that can cause adverse effects by
interfering in some way with the body’s hormones
or chemical messengers. These substances are
therefore called hormone disruptors or endocrine
disruptors, as it is the endocrine glands that secrete
the hormones. Hormones play a crucial role in
guiding normal cell differentiation in early life forms,
and so exposure to endocrine disrupting substances
in the egg or in the womb (mammals) can alter the
normal process of development. Lately, most
attention has been focused on estrogens; natural or
synthetic compounds that elicit a feminizing effect
by binding to the cellular estrogen receptor in
organisms. The interaction between an estrogenic
compound and its receptor causes a number of
Adverse effects of pesticides and chemicals
reactions and development. Environmental problems
with estrogenic compounds seem to occur primarily
in the aquatic environment, like feminization of male
fish [2]. Adult animals can also be affected, but it is
the embryonic stages that are especially vulnerable.
Exposure at this sensitive time may cause effects in
mammalian systems that are not evident until later in
life, such as effects on learning ability, behavior,
reproduction and increased susceptibility to cancer
and other diseases.
The pesticides and related chemicals originating
from human activity or agricultural farming are
discharged directly or indirectly into the receiving
waters. The presence of these chemicals in the
environment has become a global issue. Field studies
have shown that the reproduction, growth and
development of wildlife species, including
invertebrates, amphibians, reptiles, fish, birds and
mammals may have been impacted by chemicals that
interact with the endocrine system. Pesticides at low
concentrations may act as blockers of sex hormones,
causing abnormal sexual development, abnormal sex
ratios, and unusual mating behavior. Pesticides can
also interfere with other hormonal processes, such
as thyroid and its influence on bone development
[3]. This paper reviews the adverse effects of
pesticides and related chemicals on enzyme and
hormone systems in fish, amphibians and reptiles.
Effects on fish
Fish species are sensitive to enzymic and
hormone disruptors. Chronic exposure to low levels
of pesticides may have a more significant effect on
fish populations than acute poisoning. Doses of
pesticides that are not high enough to kill fish are
associated with subtle changes in behavior and
physiology that impair both survival and reproduction
[4]. Biochemical changes induced by pesticidal stress
lead to metabolic disturbances, inhibition of
important enzymes, retardation of growth and
reduction in the fecundity and longevity of the
316
organism [5]. Liver, kidney, brain and gills are the
most vulnerable organs of a fish exposed to the
medium containing any type of toxicant [6]. The fish
show restlessness, rapid body movement,
convulsions, difficulty in respiration, excess mucous
secretion, change in color, and loss of balance when
exposed to pesticides. Similar changes in behavior
are also observed in several fishes exposed to
different pesticides [7].
The Great Lakes fish are contaminated with
chlorinated organic compounds such as PCB and
dichlorodiphenyl dichloroethene, pesticides such as
mirex and dieldrin, and trace amounts of metals such
as lead and mercury [8]. Lake trout, which became
extinct in the Great Lakes in the 1950s, has been
shown to be very sensitive to dioxins and
(polychlorinated biphenyls) PCBs when exposed as
embryos. Several species of salmon introduced into
the Great Lakes have severely enlarged thyroid
glands, which is strong evidence of hormone
disruption. Salmon in the Lake Erie show a variety
of reproductive and developmental problems, for
example, early sexual development and a loss of the
typical male secondary sexual characteristics, such
as heavy protruding jaws and red coloration on the
flanks.
Some agrochemicals can indirectly affect fish
by interfering with their food supply or altering the
aquatic habitat, even when the concentrations are
too low to affect the fish directly. Other agricultural
chemicals are capable of killing salmon and other
aquatic animals directly and within a short period of
time. For example, in 1996 the herbicide acrolein
was responsible for the death of approximately
92,000 steel-head, 114 juvenile coho salmon, 19
resident rainbow trout, and thousands of non-game
fish in the Bear Creek, a tributary of the Rogue River
[3]. Several laboratory experiments show that
sublethal concentrations of agrochemicals can affect
many aspects of salmon biology, including a number
of behavioral effects [3].
317
Under experimental conditions, rainbow trout
exposed for 18-34 days to a combination of 0.05
mg/l of the organochlorine endosulfan and 0.5 mg/l
of the organophosphate disulfoton showed changes
in the ultrastructure of hepatic cells, with irregular
nuclei, and alterations to the lysozomes and rough
endoplasmic reticulum [9]. Some pesticides such as
organochlorine, organophosphates and carbamates
are known to cause morphological damage to the
fish testis. These also affect female fish in the same
way. They cause delayed oocyte development and
inhibition of steroid hormone synthesis [10].
Experimental exposure of fish to them has been
shown to depress protein values in brain, gills, muscle,
kidney and liver. In the kidney and the liver there is
evidence of significant decrease in the protein content
due to stress in elimination and also in metabolism
[11].
Interference with endocrine hormones affects
reproduction, immune function, development, and
neurological functions in several species of wild
animals. In fish, endocrine disruptors interrupt normal
development and cause male fish to have female
characteristics. These outward symptoms of
developmental disruption are accompanied by
reduced fertility and even sterility in adults, as well
as lower hatching rates and viability of offspring.
Many studies show a direct relationship between
concentrations of pesticides and related chemicals
in fish tissues and depressed hormone
concentrations. Disruption of the balance of
endocrine hormones during development of young
fish can also cause defects of the skeletal system,
resulting in deformities and stunted growth [3,12].
The common pesticide synergist piperonyl
butoxide increases carbaryl toxicity (Carbaryl is a
neurotoxic carbamate pesticide). In fish, acute
toxicity of a carbaryl- piperonyl butoxide mixture
was over 100 times that of carbaryl alone [13]. In
addition, carbaryl increases the acute toxicity of the
M. Zaheer Khan & F.C.P. Law
phenoxy herbicide 2,4-D, the insecticides rotenone
and dieldrin (an organochlorine) as well as the wood
preservative pentachlorophenol [14]. Sublethal
effects of the organophosphate insecticide
phenthoate are also synergized by carbaryl in fish,
resulting in AChE inhibition [15] and both
morphological and behavioral changes [16]. While
the toxicity of combinations of chemicals is rarely
studied, the ability of carbaryl to interact with a large
number of chemical classes is striking.
Effects on amphibians
Concern over the decline of amphibians
globally has highlighted the importance of using this
group as a bioindicator of environmental
contamination and climate change. Since 1989, there
has been a growing realization that amphibian
populations have been declining at an alarming rate.
The present data show significant declines all over
the world. Nearly 600 amphibian populations studied
in Western Europe show 53% decline beginning in
the 1950s [17]. In North and South America, 54%
and 60% of the populations, respectively, have
shown significant declines. In Australia and New
Zealand, as much as 70% of the amphibian
populations studied have declined. According to the
2004 IUCN Red List [18], there are 20 countries
with the highest number of threatened amphibians
(Table 1). The numbers of threatened species are
increasing in all taxonomic groups (Table 2) due to
environmental pollution (including pesticides and
related chemicals), habitat destruction as well as
climate change. During the year 2000 and 2004, the
number of extinctions in the fish, amphibian, reptilian,
mammalian and avian groups has increased further.
There are 338 species which are considered extinct
and another 22 species are considered extinct in the
wild (Table 3).
Adverse effects of pesticides and chemicals 318

Table 1. Amphibians are important components of

Countries with highest number of threatened aquatic habitats, especially in tropical regions of the
amphibians [18]. world [22]. The mechanisms responsible for the
Rank Country Threatened decline of amphibian populations include chemical
Species pollution from pesticides and fertilizers and global
1 Colombia 208 climate change [19]. The health of amphibians can
2 Mexico 191 suffer from exposure to pesticides [20]. Because of
3 Ecuador 163 their semipermeable skin, the development of eggs
4 Brazil 110 and larvae in water, and the position in the food web,
5 China 86 amphibians are prone to adverse effects of
6 Peru 78 waterborne and airborne pollutants in their breeding
7 Guatemala 74 and foraging habitats [21]. Pesticides may affect
8 Venezuela 68 amphibian populations in a number of ways [27];
9 India 66 they may kill individual amphibians directly [28] or
10 Costa Rica 61 indirectly through alterations in immune or
11 Madagascar 55 neurological function [29]. Pesticides may also affect
12 Honduras 53 recruitment in amphibian populations by disrupting
13 Panama 52 normal growth and development of the young or by
14 USA 51 impairing adult reproduction [27]. An extensive
15 Cameroon 50 research study conducted in Quebec, Canada, shows
16 Philippines 48 that hind limb deformities are commonly observed
17 Australia 47 in transformed bullfrogs, green frogs, northern
18 Cuba 47 leopard frogs, and American toads [23]. Deformity
19 Haiti 46 rates tend to be higher at agricultural areas, suggesting
20 Malaysia 45 that herbicides and pesticides are the likely causes.

Table 2.
Numbers of threatened species by major taxonomic group [18].

Taxonomic Number of Number of Number of Number Number

group described species threatened threatened threatened as %
species evaluated species in as % of species of species
2004 described evaluated

Fish 28,500 1,721 800 3% 46%

Amphibians 5,743 5,743 1,856 32% 32%

Reptiles 8,163 499 304 4% 61%

Mammals 5,416 4,853 1,101 20% 23%

Birds 9,917 9,917 1,213 12% 12%

319
Table 3.
Numbers of extinct (EX) and extinct in the wild (EW) species by taxonomic group in 2004 [18].
Taxonomic group Extinct (EX) Extinct in the wild (EW)
Fish 81
Amphibians 34
Reptiles 21
Mammals 73
Birds 129
Total 338
Because deformities of frogs do not always occur in
these areas, a number of other factors may be
involved, including the incidence or abundance of
certain diseases or parasites [24]. Fifteen amphibian
species have been designated as endangered,
threatened, or of special concern by the Committee
on the Status of Endangered Wildlife Canada due to
some threats including chemical contamination [24].
Amphibians are known to be vulnerable to
pesticides that are cholinesterase inhibitors [25].
Anticholinesterase pesticides function by binding with
this enzyme in animals and disrupting nervous system
activity, usually causing death by respiratory failure.
Decreased cholinesterase activity can indicate
exposure to some commonly used pesticides and
can be harmful to wild animals [26]. The
anticholinesterase effects of two other pesticides,
Lambda cyhalothrin (a pyrethroid) and
monocrotophos (an organophosphate), on Rana
cyanophlyctis (Skittering frog) have been observed
in the liver, kidney and brain. About 34.6 - 46.3%,
25.08 - 57.1% and 31.64 - 50.7% of the
cholinesterase activities in the liver, kidney and brain,
respectively are reduced following exposure to
cyhalothrin. For monocrotophos treatment,
cholinesterase decreases about 37.7 - 57.7%, 57.5
- 67.5% and 47.6 - 65.9% in the liver, kidney and
M. Zaheer Khan & F.C.P. Law
Total
12 93
01 35
01 22
04 77
04 133
22 360
brain, respectively [38]. The effect of two
pyrethroids, Lambda cyhalothrin and Permethrin, on
the cholinesterase activity of amphibian R.
cyanophlyctis and Rana tigrina have also been
compared. The cholinesterase activities of cyhalothrin
treated frogs are decreased 34.6 - 46.3%, 25.08 -
57.1% in the liver and kidney. Permethrin treatment
decreases cholinesterase activity 23 - 29% and 6.76
- 35% in the liver and kidney, respectively [39]. Total
protein content also decreases in non-target
amphibians after pesticide treatment, indicating
pesticide-produced changes in the biochemical
systems of non-target organisms [40]. The brain
cholinesterase activity of Rana cyanophlyctis
decreases upto 4.10 and 13.84 % under the effect
of sandaphos and 5.16 and 23.28% under the effect
of b-cypermethrin, respectively [41].
Some pesticides, herbicides, and nematocides
are documented to have endocrine-disrupting effects
[30]. To date, there are no reports linking endocrine
dysfunction with amphibian malformations. However,
it is well established that pesticides and related
chemicals can be major ecological threats to fish and
aquatic wildlife by diminishing productivity and
fecundity [30]. The normal growth and development
of amphibian larvae rely on functional and
uncontaminated aquatic systems. Water sources are
Adverse effects of pesticides and chemicals
particularly at risk to contamination by pesticides
because of the accumulation and distribution of
contaminating substances in sediments of rivers,
lakes, and ponds. Potential sources of EDCs that
impact bodies of water include municipal sewage
[31], and agricultural runoff (pesticides and
herbicides) [32]. Thus, the EDCs that accumulate in
aquatic systems may adversely affect amphibian
reproductive processes.
Exposure of amphibians to dimethoate,
carbofuran and chlorpyifos can alter vitamin A levels
[33] and reduce melanogenesis [34]. Carbaryl, a
short-lived carbamate that acts through
acetylcholinesterase inhibition, may serve as a model
chemical for neurotoxins (i.e., carbamates and
organophosphates). The effects of sublethal
concentrations are more relevant to amphibian
communities because they may directly affect time
of and size to metamorphosis, or indirectly affect
survival [35].
Some studies have reported that the tadpole
stage of Rana spp. is sensitive to herbicides, and
various types of deformities in the tadpoles serve as
possible indicators of such sensitivity. Tadpoles are
also highly sensitive to organochlorine pesticides, and
toxic effects of these pesticides are evident during
metamorphosis [29], a period of marked endocrine
change and reduction. In Minnesota, USA, some
pesticides or their degradation products have been
detected in water and sediment samples in very small
quantities [36]. Despite the current documentation
of amphibian declines and malformations, there are
only few reports on the use of amphibians as models
for abnormalities of reproductive processes by
exposure to EDCs. In one study, the interactions of
gonadal steroids and pesticides (DDT, DDE) on
gonoduct growth in larval tiger salamanders,
Ambystoma tigrinum, were examined [37]. The
salamanders were immersed in a solution of DDE,
DDT, or injected with estradiol or dihydro-
testosterone. Essentially all the compounds tested
320
had some adverse effect on the gonoduct growth in
this species of salamanders.
Effects on reptiles
Very rapidly deteriorating status of freshwater
turtles and tortoises in Southeast Asia has resulted
in an increasing number of these species being listed
as threatened in the IUCN Red List; globally 42%
of turtle and tortoise species are threatened [18].
The decline in the population of alligator in the Lake
Apopka, Florida (USA), is contaminated by
organochlorine pesticides that emanate from a
chemical spill. Here, a number of disturbing
abnormalities were recorded in hatchlings and
juvenile alligators, including modifications of enzyme
activity, concentrations of sex hormones, abnormal
ovarian morphology and unusually small phalluses
[42,43]. Because these chemicals are known to be
weak androgen receptors, the hypothesis that the
individual and the population level effects observed
in the alligators are due to chemical disruption of
endocrine function seems reasonable [44].
The common snapping turtle (Chelydra
serpentina) is the largest freshwater turtle occurring
in Canada. Snapping turtle eggs from the Great
Lakes contain high concentrations of fat-soluble
contaminants which are absorbed while food is being
digested. These include PCBs, dioxins, furans and
organochlorine pesticides. Abnormal development,
such as incidence of unhatched eggs or deformed
animals, occurs at the highest rates in the sites which
are the most contaminated [45]. In addition, a
correlation between contaminated eggs and reduced
developmental success has also been indicated [46].
The anticholinesterase effects of the
phytopesticide, biosal (neem based formulation), on
Indian garden lizard (Calotes versicolor) have been
observed in the kidney and liver. About 13.60 - 18%
and 39.52 - 52.61% of the cholinesterase activities
in the kidney and liver are reduced following
321
exposure to biosal [47].
Conclusion
This review shows that pesticides and related
chemicals are capable of blocking the action of
hormones in fish, amphibians and reptiles and causing
reproductive dysfunction and abnormal
development. They act on target tissues through
hormone receptors or nonreceptors, may influence
hormone secretion or its clearance from the body.
In the last years, a number of deformed frogs have
been found in the eastern US and Canada. The cause
of mass deformities of transforming frogs remains
elusive, but various factors have been implicated,
including pesticides and related chemicals. The role
of pesticides and related chemicals in amphibian
malformations may be of concern due to the high
deformity rates associated with sites where
agricultural chemicals have been used. These
chemicals can be a major threat to fish, amphibians
and reptiles, and aquatic environment by diminishing
productivity and fecundity. Further research is
needed to evaluate the effectiveness of alternative
pesticides and related chemicals to reduce the effects
on fish, amphibian and reptilian populations. Globally,
studies need to simultaneously consider the benefits
to both agricultural and conservation communities;
scientists from both communities should provide
input to make realistic and informed decisions about
the protection and conservation of aquatic
biodiversity within agricultural landscapes.
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